PROJECT SUMMARY Accurate partitioning of the replicated genome during cell division is essential for the normal development of all organisms. Chromosome segregation errors lead to aneuploidy, a hallmark of cancer and a common origin of birth defects. The chromosome segregation machinery is also an important target in cancer therapy and elevated rates of chromosome missegregation, observed in many cancers, are associated with therapeutic resistance. Thus, elucidating the mechanisms ensuring accurate chromosome segregation has the potential to contribute to understanding the genesis of cancer and guide the development of new therapeutic strategies. A central player in chromosome segregation is the kinetochore, the machine that assembles on mitotic chromosomes to interface with spindle microtubules. The mechanics of this interface are integrated with regulatory mechanisms that modulate the strength of kinetochore-microtubule attachments, correct attachment errors, and prevent cell cycle progression until all chromosomes are connected to the spindle. Mechanical and regulatory functions are coordinated at the kinetochore by the conserved Knl1 complex/Mis12 complex /Ndc80 complex (KMN) protein network. While substantial progress has been made in characterizing the kinases that control the mechanical and regulatory aspects of chromosome segregation, understanding of the conserved opposing kinetochore-localized phosphatase, protein phosphatase 1 (PP1c) has lagged behind. Aims 1 and 2 address this gap by defining the mechanisms that control kinetochore localization, activity and substrate specificity of PP1c, in addition to determining how kinetochore-docked PP1c controls anaphase onset and regulates microtubule attachments. To ensure accurate chromosome segregation, chromosomes must achieve bi-orientation on the spindle, the state in which sister chromatids are exclusively connected to opposite spindle poles. Widely studied pathways such as the spindle checkpoint and error correction by Aurora kinases act to ensure bi-orientation. We defined a pathway that acts after bi-orientation to ensure accurate segregation by stabilizing properly oriented kinetochore-microtubule attachments. Aim 2 also focuses on understanding the mechanistic basis of this pathway, which involves coordination between the conserved kinetochore-localized microtubule-binding Ndc80 and Ska complexes and potential regulation of their coordination by PP1c. Finally, Aim 3 pursues two new directions that emerged from our working in a multicellular genetic model. The first is based on our discovery that the KMN network has an important non-mitotic role in formation of the nervous system during embryogenesis. The second is based on our surprising finding that the critical organismal function of conserved spindle checkpoint components is kinetochore-independent promotion of mitotic entry in the germline. The work proposed in this final aim will define new and unexpected biological functions for well- studied chromosome segregation machinery and has the potential to influence strategies directed at therapeutic targeting of this machinery in cancer.